High-Throughput Assays for Inhibitors of Understudied Bacterial Proteases Bacteria that colonize the human mucosa can become virulent and invasive, causing chronic and acute diseases including drug-resistant gonorrhea, pneumonia, and meningitis. New drugs targeting colonization and invasion would be welcome additions to the antibacterial arsenal, but interactions among bacterial factors, host tissues, and host immune defenses are poorly understood. For instance, immunoglobulin A1 (IgA1) represents an important barrier to colonization of mucosal surfaces, and pathogenic strains of several Gram- positive and Gram-negative bacteria produce proteases that cleave IgA1. These include Neisserial, Haemophilus, and Streptococcus strains that cause chronic ear infections (otitis media), bronchitis, drug- resistant gonorrhea, bacterial pneumonia and bacterial meningitis. Despite a large body of evidence that IgAPs are virulence factors with diverse functions, they have been vastly understudied because there were few assays for measuring their activity and no selective inhibitors. Importantly, the potential for targeting IgAPs as an ani-virulence therapy remains untested, and will remain so until selective inhibitors are identified. The Kritzer lab uses synthesis, biophysical chemistry, and cell biology to develop inhibitors of difficult-to-target proteins. Recently, we designed fluorescence probes to quantitate IgAP activity from diverse human pathogens. Despite forty years of research into IgAP biology, these probes are the first and only of their kind and enable the first-ever high-throughput screens for IgAP inhibitors. The goal of this R01 proposal is to leverage these probes into high-throughput screens that will deliver selective inhibitors of IgAPs. We will accomplish this goal by: testing additional substrate sequences, developing the substrates into fluorescent probes, incorporating the probes into HTS assays for IgAP inhibitors, performing pilot screens, and testing hits in follow-up assays that measure their effects in vitro and on live bacteria. We have partnered with the Broad Institute to ensure smooth transitions from probe development to pilot assays to a full screening campaign. The assays developed in this project will be of general use to microbiologists and immunologists for quantifying IgAP activity. They could also find clinical applications in the diagnosis of bacterial meningitis and other infections. Most importantly, the inhibitors developed from high-throughput screening will allow us and others to investigate host-pathogen interactions involving IgAPs. The inhibitors will also represent starting points for evaluating IgAPs as drug targets for treating urgent infectious disease threats, including drug- resistant gonorrhea, bacterial pneumonia, and bacterial meningitis.